Principal Investigator/Program Director (Last, first, middle): Zarour, Hassane, M. PROJECT SUMMARY/ABSTRACT CD4+T cells play critical roles in the induction and maintenance of anti-tumor responses. In order to develop and optimize immunotherapeutic approaches to stimulate human anti-tumor CD4+ T cells in vivo, we have previously identified a number of promiscuous MHC class II-restricted epitopes from cancer-germline antigens (CAGs), including NY-ESO-1 and LAGE-1, melanocyte-lineage antigens (Melan-A/MART-1) and most recently overexpressed or universal antigens (Survivin). We have generated antigen-specific CD4+ T cells recognizing these epitopes and characterized their cytokine profile production to define Th1-type and Th2/Th0-type CD4+ cells. We also have engineered modified peptides to better identify the amino acids (aa) involved in peptide- MHC binding or TCR contacts. Most recently, we have developed assays to study the functions of the CAG- specific CD4+ T cells derived from the peripheral blood lymphocytes (PBLs) of patients with melanoma. Our preliminary data have suggested that circulating CAG-specific CD4+ T cells may not only be Th1-type helper CD4+ T cells but also CD4+ regulatory T cells. It is thus possible that active immunosuppression by these tumor antigen-specific CD4+ T cells may represent a major obstacle to the successful vaccination with T- helper epitopes. In the current competitive renewal, we propose to address this important issue. The rationale for our proposed research project is several fold and can be stated as follows: 1) T-helper epitopes may stimulate not only antigen-specific T-helper type CD4+ T cells but also CD4+ regulatory T cells present in the tumor microenvironment and circulating PBLs; 2) Tumor-antigen-specific regulatory cells may act on autologous B cells to inhibit tumor antigen-specific antibody production; 3) strategies based on optimizing strength of TCR stimuli and costimuli will inhibit regulatory functions and promote T-helper functions. Our long- term goal is to develop the knowledge required for the induction of CAG-specific T-helper responses in vivo in patients with advanced melanoma.Our Specific Aims are: 1) To generate and characterize tumor antigen- specific CD4+ regulatory T cells from PBLs of melanoma patients and normal donors; 2) To assess the impact of tumor-antigen-specific regulatory T cells on autologous B cells; and 3) To define strategies to preferentially stimulate tumor antigen-specific T-helper CD4+ T cells instead of regulatory T cells. Project Description Page 6